The heterotrophic dinoflagellates Oxyrrhis marina and Oxyrrhis maritima in Korean waters : taxonomy, physiology, distribution, and production of useful materials

Abstract

The genus Oxyrrhis is a heterotrophic dinoflagellate (HTD) which is often found in diverse marine environments such as coastal waters, tidal pools, and salterns. Oxyrrhis spp. have received much attention owing to their ecological and industrial importance. They play diverse roles such as prey for other protists or metazoans and predators for phytoplankton and bacteria in marine ecosystems. Furthermore, they produce several useful materials such as docosahexaenoic acid (DHA), eicosapentaenoic acid (EPA), and anti-inflammatory materials. For a long time, Oxyrrhis marina was considered a single species in the genus Oxyrrhis, after Oxyrrhis marina, O. maritima, O. phaeocysticola, and O. tentaculifera had been merged to O. marina. However, in 2011, O. maritima was separated from O. marina due to considerable difference in their genetic characterizations, although morphological difference between these two species was not significant. Although morphological and genetic characterizations are still important proxy of genomic characterizations, eco-physiological characterizations of these two species may provide a clue in solving the question whether they are the same or different species. To the best of our knowledges, many studies on eco-physiological characterizations of O. marina, but no studies on those of O. maritima have been reported until date. Therefore, I investigated the taxonomy and eco-physiology of O. marina and O. maritima using four O. marina strains from the coastal waters off Gunsan, Masan, Shiwha, and Karorim, Korea and two O. maritima strains were isolated from littoral tidepool waters in Jeju Island, Korea.

Difference among the sequences of the small subunit (SSU) rDNA of the four strains of O. marina was 0.3-3.5%, whereas that between two strains of O. maritima was only 0.3%. However, the difference between the sequences of SSU rDNA of O. marina and O. maritima was 17.3-18.1%. In the phylogenetic trees based on the SSU rDNA of dinoflagellates, the clade of all the four strains of O. marina was clearly divergent from that of the two strains of O. maritima. Furthermore, the O. marina strains belonged to the Lineage I (Clade 1 and 2), whereas the O. maritima strains belonged to the Lineage II (Clade 4). Thus, the results of this study suggest that O. marina and O. maritima are different species. Moreover, I found that the Korean strains of O. marina were splitted to two clades. In the present study, I measured the growth rates of O. marina and O. maritima under various water temperatures (5-36 ℃), salinities (2-90), and light intensities (0-100 µE m-2·s-1). The strains of O. maritima grew at the temperatures of > 35 ℃, but O. marina did not grow. Furthermore, the optimal temperature supporting the maximum growth rate of O. marina was 25 ℃, while that of O. maritima was 30 ℃. Moreover, O. marina did not survive at a salinity of < 4, while O. maritima survived at a salinity of 2. In addition, at a salinity of 50, the growth rate of O. maritima (0.67 d-1) was higher than that of O. marina (0.58 d-1). Thus, O. maritima has wider ranges of temperature and salinity than for O. marina. Thus, theses two Oxyrrhis species may have different eco-physiological characterizations.

Using direct counting, real-time polymerase chain reaction (qPCR), and digital PCR (dPCR), I measured the abundances of O. marina and O. maritima in the waters collected from the 29 stations along the Korean coasts in January, March, May, July, August, October, and December in 2016 and also from tide-pools and salterns located in Taean and Jeju Island in June and September in 2016. In the dPCR method, O. maritima (177 ± 9.2 copies per cell) has copy numbers greater than O. marina (148 ± 6.9 copies per cell), even though O. maritima cell size is smaller than that of O. marina. In the qPCR and dPCR methods, the abundance of O. marina was rarely detected in Korean coastal waters, whereas that of O. maritima was frequently detected in southern Korea coastal waters in 2016. In addition, two species had much higher abundances in the tide pool or saltern (highest abundances : O. marina, 7,490 cells ml-1

O. maritima, 3,700 cells ml-1) than that in the coastal waters (highest abundances : O. marina, 39 cells ml-1

O. maritima, 3 cells ml-1). Therefore, the abundances of O. marina and O. maritima were markedly affected by their habitats.

Moreover, I investigated new compounds from the massive culture of O. marina. O. marina can grow fast and is known to produce high amounts of useful lipids, such as docosahexaenoic acid (DHA). I isolated two new compounds, a trioxilin and a sulfoquinovosyl diacylglycerol (SQDG) from a massive culture of O. marina, which was cultivated by feeding on dried yeast. The complete structures of these compounds were determined using nuclear magnetic resonance (NMR) spectroscopy and chemical reactions. The trioxilin was identified as (4Z,8E,13Z,16Z,19Z)-7(S),10(S),11(S) -trihydroxydocosapentaenoic acid, and the SQDG was identified as (2S)-1-O-hexadecanosy–2–O–docosahexaenoyl–3-O-(6-sulfo-α-d-quinovopyranosyl)-glycerol by a combination of NMR spectra, mass, and chemical analyses. These two compounds were associated with DHA, which is a major component of O. marina. The two isolated compounds showed significant anti-inhibitory activity in lipopolysaccharide-induced RAW264.7 cells. The second compound showed no cytotoxicity against hepatocarcinoma (HepG2), neuroblastoma (Neuro-2a), and colon cancer (HCT-116) cells, while weak cytotoxicity of the first compound 1 against Neuro-2a cells was observed.

Furthermore, I investigated a new massive culture method which can produce high levels of DHA from O. marina. O. marina contains high levels of DHA when fed on diverse algal prey. However, large-scale culturing of algal prey species is not easy and requires a large amount of budget. Therefore, a more easily cultivable and low-cost prey is required. I found out that dried yeast was a strong candidate for an alternative prey and explored the fatty acid composition and DHA production of O. marina fed on dried yeast. In addition, I compared these results with those of O. marina fed on two algal prey species: the phototrophic dinoflagellate Amphidinium carterae and chlorophyte Chlorella sp. powder. O. marina fed on dried yeast, which does not contain DHA, produced the high levels of DHA, in as those fed on DHA-containing A. carterae. This indicates that O. marina is likely to produce DHA regardless of the prey items. Furthermore, the DHA content (and portion of total fatty acid methyl esters) of O. marina satiated with dried yeast was 52.40 pg per cell and 25.9%), respectively. These values were considerably greater than those of O. marina fed on other algal prey species. Therefore, dried yeast is a more easily obtainable and cost-effective prey than is conventional algal prey for use in the production of DHA by O. marina.

In summary, I established clonal cultures of six different strains of Oxyrrhis species and identified them as Oxyrrhis marina and O. maritima based on morphological and molecular taxonomic analyses. In addition, I investigated their ecological and physiological response to diverse environmental parameters and distribution in Korean waters based on their taxonomic characteristics. Moreover, I investigated useful new compounds of O. marina and developed a new cost-effective mass culture method for this species. The finding of this study will contribute to the improvement of our understanding of the role of Oxyrrhis species in marine waters and commercialize useful materials from algal species.